Our general aim is to establish a mechanistic connection between the ability of mechanical forces in intact tissue to alter conformation of an ECM protein, fibronectin (FN), and subsequent signaling events that result in changes in arteriolar diameter. Our published work (REF) established that FN signaling contributes to the dilation produced by muscle contraction, thus identifying a new mechanism regulating small resistance arterioles. We will use FN-mimetic peptides that we have constructed to explore arteriolar responses using confocal intravital microscopy of intact tissues in anesthetized WT and knockout animals, complemented by studies in isolated cells. Specific Aim 1 will determine the roles of HSPG- and integrin-ligation in maintenance of vascular tone and in arteriolar dilation. Hypothesis Part I: Active contraction of skeletal muscle of intact, adult animals transiently exposes the matricryptic III-1 site in the surrounding ECM FN. Subsequent ligation of HSPGs on cell surfaces with III-1H triggers local vasodilation by a 21 integrin- dependent mechanism. Part II: Under resting conditions, a basal level of ligation of HSPGs on cell surfaces contributes to maintenance of resting vascular tone. Specific Aim 2 will determine the role of eNOS, nNOS, caveolin and endothelial cell Ca2+ in maintenance of resting tone and in arteriolar dilation. Hypothesis: Part I: Active contraction of skeletal muscle of intact, adult animals transiently exposes the matricryptic III-1 site in the surrounding ECM FN. Subsequent ligation of HSPGs on cell surfaces with III-1H triggers local vasodilation by a caveolin- and NO-dependent mechanism. Part II: Under resting conditions, a basal level of ligation of HSPGs on cell surfaces contributes to maintenance of resting vascular tone via NO-dependent mechanisms. Specific Aim 3 will identify the role of Src signaling in FN-dependent responses. Hypothesis: Ligation of the FNIII-1H site on ECM FN generates NO via a Src kinase-dependent mechanism. Specific Aim 4 will visualize changes in ECM FN conformation in response to skeletal muscle contraction and determine how tissue strain in response to mechanical force exposes the FNIII-1 matricryptic site in connective tissue. Hypothesis: Tissue strain in response to skeletal muscle contraction alters the conformation of ECM FN fibrils and exposes a matricryptic site in FNIII-1. This project is a critical step towards understanding how mechanical forces in the tissue affect FN conformation and hence vascular responses, under normal and pathological conditions, for example the integrated response to exercise, or the changes in peripheral vascular function associated with aging, where changes in ECM protein composition are documented. This proposal will bring together a unique interdisciplinary approach combining expertise in FN matrix biology and microvascular function to use a novel paradigm addressing a key question in vascular biology, that of mechanisms for transduction of mechanical signals into vascular responses. PUBLIC HEALTH RELEVANCE: This project explores how the connective tissue that surrounds arterioles can generate signals that modify responses in these vessels. These vessels are the ones that primarily regulate peripheral blood flow, hence it is of considerable significance to understand how changes in the connective tissue proteins can alter how the blood flow is regulated. One example of where this may be very significant for human health is in aging populations, where peripheral blood vessel function often deteriorates, but the mechanisms are unknown;our work raises the possibility that some of this change in vascular function may relate to changes in signals from connective tissue proteins.